The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of vascular effects in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. In recent studies we have infused nitrite into the brachial arteries of normal human volunteers and have shown that this increases blood flow, suggesting that nitrite could function physiologically as a source of NO and could be used pharmacologically. However, we find that the effects of nitrite infusion, both on vascular properties and on methemoglobin formation, are relatively long lasting and suggest partition of the nitrite into various tissues. We find that in vitro deoxyerythrocytes and nitrite cause aortic ring preparations to dilate, suggesting a mechanism of nitrite activation by deoxyheme proteins. We also find that nitrite inhalation in hypoxic newborn sheep lead to decreased pulmonary artery pressures and exhalation of NO;nitrite infusions in these animals leads to decreases in mean arterial blood pressure. We are currently studying the formation and compartmentalization of nitrite in the blood, in erythrocytes in particular, and whether nitrite levels may be a marker of cardiovascular risk in humans. These studies are designed to allow us to initiate nitrite infusions in normal human subjects and those with a variety of ischemic (including sickle cell anemia) diseases. We have shown that the maximum production of NO from nitrite occurs near the p50 of hemoglobin and is dependent on the allosteric conformation of hemoglobin. We have also developed methods to measure nitrite levels precisely in human blood and have found that most of blood nitrite is contained in the red cells. In the recent reporting periods, we have measured the uptake of nitrite into the human red cell as a function of oxygen tension, pH, temperature and other variables. Such uptake appears to be an important factor controlling the overall rates of conversion of nitrite to NO intravascularly. Further, we have shown that dehydroascorbic acid in red cells can catalyze the oxidation of iron-nitrosylhemoglobin (NbNO) to release NO and that this NO is then converted to nitrite but not nitrate intracellularly. This reaction may be the major mechanism for the formation of red cell nitrite, which we believe is one of the major storage sites for bioactive NO in the body, and has led us to develop a model of the interaction of the ascorbic acid/dehyroascorbic acid and the NO/nitrite cycles inside the erythrocyte. We have also examined the hypothesis that in hypoxia cell-free hemoglobin could serve as an additional endocrine source of NO. In this study, we constructed a multicellular model to characterize the amount of NO delivered by the reaction of nitrite with both intraerythrocytic and cell-free hemoglobin and find that levels of NO production by this mechanism may be quite significant, comparable to production by the vascular wall itself. We have also examined in detail the reaction mechanism of nitrite with oxy- and deoxy-hemoglobin and find that at conditions likely to obtain physiologically and pharmacologically-especially with regard to the ratio of nitrite ions to hemoglobin molecules-the formation of free radicals (including that of the ferryl-form of hemoglobin) is not likely to occur to a major extent. This is important because the formation of free radicals had been of concern with regard to the development of nitrite (or NO) pharmacology. We believe that the above studies should contribute to our understanding of the role of the human erythrocyte in modulating NO bioactivity, especially via a nitrite intermediate, and also facilitate the development of nitrite as a useful drug for cardiovascular pathology. In recent work we have been investigating changes in NO-related species during red blood cell storage to ascertain whether these contribute to the complications of blood, especially red cell, transfusion know as the "storage lesion." We find, as expected from our previous work that nitrite levels fall rapidly after venisection but then, surprisingly level off at about 1/4 of the initial value for up to 42 days. We find no evidence of other relevant NO changes and are now investigating whether the nitrite changes contribute to red cell-induced pathology and, equally importantly, the mechanism of control and stabilization of red cell nitrite levels. In the last year we have shown that at physiological nitrite concentrations we can generate enough NO to inhibit platelet aggregation;we are now working on the physiological and pharmacological implications of these results, which appear to involve interactions of nitrite with circulating red blood cells and may contribute to physiological and pathophysiological modulation of platelet reactivity in the circulation. This work is closely related to that described in the report on potential nitrite therapeutics and the levels of nitrite in platelets during storage in vitro.